For the FBI, the case of the anthrax killer is an investigation that never seems to end.

Agents thought they had solved the puzzle last year when they pinned the 2001 attacks on a deceased Fort Detrick scientist. But yet another new wrinkle emerged Tuesday, with a panel of prominent scientists casting doubt on key FBI scientific evidence.

A report from the National Research Council questioned the strength of genetic testing that the government said had conclusively linked the anthrax-infested letters that killed five people to a flask of lethal bacteria belonging to Bruce E. Ivins. The Justice Department closed the probe last year after concluding that Ivins, an Army researcher who killed himself in 2008 as investigators closed in on him, had single-handedly prepared and mailed the anthrax spores, terrorizing the nation just after the Sept. 11, 2001, attacks.

It was another turn in a tumultous investigation that initially focused on the wrong man, then fingered Ivins even as some of his fellow scientists expressed doubts about his guilt. Those doubts were reignited Tuesday, with three members of Congress calling for a review of the FBI's investigation along the lines of that by the independent commission that probed Sept. 11.

"For years, the FBI has claimed scientific evidence for its conclusion that anthrax spores found in the letters were linked to the anthrax bacteria found in Dr. Ivins' lab," said Sen. Charles E. Grassley (R-Iowa). The report "shows that the science is not necessarily a slam-dunk. There are no more excuses for avoiding an independent review."

For federal law enforcement officials, who conducted a global investigation that included testing a suspected al-Qaeda laboratory and the remains of some Sept. 11 hijackers for anthrax, the developments caused palpable frustration. The report makes no judgment about Ivins's guilt or innocence, and officials stood behind their contention that he was the anthrax killer, citing what they called overwhelming evidence.

And officials criticized what they characterized as the science-paper type approach of the report, which took aim at some FBI methods it said were "not optimal" and lacked scientific rigor. They said they balanced proper science with the demands of a fast-moving criminal investigation.

"It's somewhat disengenuous that they can use the word 'theoretical,' " said one investigator involved in the case, who spoke on the condition of anonymity to be candid about the report's findings. "They're talking about hypotheticals. We didn't have that luxury. We were trying to solve a crime, and we didn't know if the mailer would strike again."

The spore-laden letters sent to news media and two U.S. Senate offices in September and October 2001 sickened 17 people and triggered a massive FBI probe that has included missteps, including the public naming of a "person of interest" who was never charged.

In a report released last year, the Justice Department concluded that Ivins hatched the anthrax-by-mail scheme in hopes of creating a scare because he was concerned about the fate of an anthrax vaccine program he had helped create. On Tuesday, officials again pointed to evidience linking Ivins to the attacks, including e-mails and recorded conversations showing an increasingly agitated Ivins seeking to implicate colleagues while misleading investigators about his ability to make the powder.

But Tuesday's report questioned a critical piece of evidence: the link between the anthrax spores in a flask - labeled RMR-1029 - stored in Ivins's lab at Fort Detrick, Md., and the anthrax from the attacks. The Justice Department report concluded that Ivins's collection of anthrax spores, which he had called his "ultimate creation," was the "parent material" for the anthrax used in the mailings.

"The scientific link between the letter material and flask number RMR-1029 is not as conclusive as stated in the DOJ Investigative Summary," said the $1.1 million report, which was commissioned by the FBI. The document added, however, that the "genetic evidence is consistent with and supports an association between the RMR-1029 flask" and the anthrax used in the attacks.

The 190-page document by the research arm of the National Academy of Sciences praised the FBI's energetic pursuit of emerging science in the investigation. But it offered another possible explanation for the apparent link between the letters and the Ivins flask: that some of the mutations identified in the letters could have arisen independently, through a process known as "parallel evolution."

The report said this possibility "was not rigorously explored" by the FBI.

In a joint statement, the FBI and Justice Department said, "The FBI has long maintained that while science played a significant role, it was the totality of the investigative process that determined the outcome of the anthrax case . . . rarely does science alone solve an investigation.''

And a federal official involved in the investigation said the government was satisfied that its science would have met the standard of proof in federal court, which is to a reasonable degree of scientific certainty. "The standard is not beyond all doubt," the official said.

The report reignited a debate that has simmered among some scientists and others who have questioned the strength of the FBI's evidence against Ivins.

"This shows what we've been saying all along: that it was all supposition based on conjecture based on guesswork, without any proof whatsoever," said Paul Kemp, an attorney for Ivins.

Paul S. Keim, director of the Center for Microbial Genetics and Genomics at Northern Arizona University, who worked with the FBI on the investigation, called the report "a qualified endorsement of the FBI's approach.''

The report endorsed a key FBI conclusion: that the anthrax spores used in the mailings had not been altered, genetically or chemically. That appeared to rule out the possibility that the spores were "weaponized" or manipulated to make them more deadly. Some scientists have pointed to oddly elevated levels of silicon in the spores as an indication that the deadly powder was enhanced by someone with knowledge of advanced bioweapons techniques.

And the report reveals that the FBI and intelligence officers collected samples from an overseas site "because of information about efforts by al-Qaeda to develop an anthrax program.''

The report said that the tests turned out to be negative but that the evidence was inconsistent, and it called for further review. Federal law enforcement officials said they thoroughly investigated the possibility of terrorist involvement in the anthrax atttacks and are certain there was none.

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